DESCRIPTION: (Applicant's abstract) Functional analysis of genomic sequences requires that mutations be created and the phenotypic consequences of the mutations assessed. In yeast, targeted mutagenesis is so proficient that gene knockouts can easily be constructed by transformation with mutating gene fragments. In mammalian cells, however, random integrations of exogenous DNA predominate, making gene knockouts laborious to construct. Current methods of mutagenesis do not allow high throughput functional analyses of cloned genes. We propose to explore a highly novel method for the introduction of targeted mutations into the mouse genome. The approach centers on the ability to introduce double-strand breaks (DSBs) at targeted locations in chromosomal DNA and the potential of mammalian cells to mutagenically repair these breaks. Mutagenic repair of DSBs in cultured cells can occur by either homologous recombination or nonhomologous endjoining mechanisms. In terms of recombination, DSB repair can stimulate gene targeting lOOOX. We plan in this proposal to determine if a DSB in the chromosome of fertilized mouse eggs will promote mutagenic repair of DSBs in the same manner as seen in tissue culture cells. In this first aim we plan to use an already defined system to study DSB repair: cleavage by a rare-cutting, site-specific endonuclease. To apply mutagenic repair of DSBs to any genomic sequence, we propose to use generalized chromosome cleavage reagents for introducing DSBs into targeted locations in the genome. These reagents rely on the pairing of an oligonucleotide to its homologous genomic sequence. Fertilized mouse eggs are utilized for exploring this process, since, after injection, they can be returned to foster mothers to give rise to mice with the targeted mutation. The co-opting of cellular DSB repair processes holds promise for generating a high throughput method for analyzing the functions of genes in mammalian genomes. By using fertilized mouse eggs and returning these fertilized mouse eggs and returning these embryos to foster mothers to produce mutant mice, the totality of the contribution of a particular gene to development and adult function can be assessed.